Integrin alpha v beta 6 targeting stapled peptides

Abstract

The invention discloses an integrin alpha v beta 6 targeting stapled peptide. Specifically, the present application discloses a peptide comprising or consisting of a peptide sequence having a first amino acid residue substitution and a second amino acid residue substitution relative to the sequence of SEQ ID NO: 28 wherein the first amino acid residue substitution is located at a position corresponding to position 11 or 12 of SEQ ID NO: 28, and the second amino acid residue substitution is located at a position corresponding to position 12 of SEQ ID NO: 28. The second amino acid residue substitution is located at a position corresponding to position 6, 14, or 15 of SEQ ID NO: 28, and the first side chain of the first amino acid residue substitution is crosslinked with the second side chain of the second amino acid residue substitution. Uses of the peptides of the application are also disclosed.

Description

Technical Field

[0001] This application relates to the field of biochemistry, and more particularly to an integrin αvβ6-targeting stapled peptide. Background Art

[0002] Biological functions of the integrin family

[0003] Integrins are one of the important members of the cell adhesion molecule family. They are a group of transmembrane glycoprotein receptors that are widely distributed on the cell surface. Its main function is to mediate the adhesion between cells and between cells and the extracellular matrix, and to mediate the bidirectional signal transduction between cells and the extracellular matrix, playing an important regulatory role in cell adhesion, proliferation, metastasis, and apoptosis. When integrins bind to extracellular ligands, intracellular signals are generated, thereby triggering various intracellular pathways. At the same time, the function of integrins can also be regulated by intracellular signals because they can serve as a link between the extracellular matrix and the actin filaments of the intracellular cytoskeleton through various actin-binding proteins. Integrins are transmembrane heterodimeric glycoproteins composed of α subunits and β subunits through non-covalent bonds. Currently, 18 α subunits and 8 β subunits have been discovered, which can form 24 different integrin receptors.

[0004] The integrin αv subunit can form heterodimers with β1, β3, β5, β6, and β8 subunits, while the β6 subunit only binds to the αv subunit. Both subunits αv and β6 contain three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. Among them, the β6 subunit contains three highly conserved sequence regions, cyto 1, cyto2, and cyto 3, which are domains necessary for binding to many cell signaling molecules and other cellular proteins. Different parts of the integrin structure have their own functions, which determine its multiple regulatory functions in tumorigenesis. For example, the extracellular domain and transmembrane domain are involved in the activation of transforming growth factor β (TGF-β), cell-cell adhesion, and epithelial-mesenchymal transition (EMT) of cells, while the intracellular domain affects cell proliferation, the production of matrix metalloproteinases (MMPS), cell migration, and survival through its 11 carboxyl-terminal amino acids.

[0005] RGD sequence ligand

[0006] Different types of integrins exhibit different ligand recognition patterns and are expressed on different cell types, which allows integrins to be classified into different types according to their ligand specificity, such as laminin-binding integrins, collagen-binding integrins, leukocyte integrins, and RGD-specific binding integrins, etc.

[0007] In the 1980s, a highly conserved integrin recognition motif consisting of three amino acids, arginine (Arg), glycine (Gly), and aspartic acid (Asp), was discovered in fibronectin. Subsequently, this conserved sequence was also found in other extracellular matrix proteins such as vitronectin, laminin, and osteopontin. Currently, there are a total of 8 different types of integrins that have an affinity for ligands containing the Arg-Gly-Asp (RGD) sequence, including αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α8β1, and αIIβ3. The specific recognition mechanism of different integrins for RGD sequence-containing ligands is very complex and mainly depends on the spatial conformation when the RGD sequence-containing ligand binds to the integrin recognition, and can be changed by altering the amino acid residues on both sides of the RGD motif within the ligand. In recent years, more and more disease diagnosis and treatment studies have focused on integrin targets, and research on its RGD ligand sequence and various targeting peptides and peptidomimetic small molecule ligands has also become increasingly extensive.

[0008] Integrins contain multiple low-affinity divalent cation binding sites in their structure, which have an important impact on the function and binding of integrins. These divalent cations (including Mg 2+ , Ca 2+ and Mn 2+ ) can promote or inhibit ligand binding and can also change the ligand specificity of integrins. A "displacement hypothesis" has been proposed, that is, ligands containing the RGD sequence interact with the divalent cations that bind to the receptor, making the interaction between the ligand and the integrin stable before the divalent ions are displaced by the RGD ligand.

[0009] αvβ6

[0010] αvβ6 is the only heterodimeric integrin formed by the participation of the β6 subunit. Integrin αvβ6 was first discovered in the 1990s. Different from most integrins, integrin αvβ6 is not expressed or weakly expressed in healthy adult epithelia, but its expression level is significantly upregulated in physiological or pathological processes such as malignant tumors, wound healing, fibrosis, and inflammation. This upregulation itself makes integrin αvβ6 a very attractive target in the diagnosis and treatment applications of various diseases.

[0011] It has been reported that during embryonic development, integrin αvβ6 is expressed in specific types of epithelial cells in the kidney, lung, and skin, while it is mostly undetectable in normal adults. In the past decade, the relationship between integrin αvβ6 and cancer has attracted increasing attention because a large number of studies have shown that the expression of integrin αvβ6 is significantly upregulated in many tumors, including pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, breast cancer, gastric cancer, oral squamous cell carcinoma, cholangiocarcinoma, etc.

[0012] In addition to its role in various cancers, during the process of fibrosis, the expression of αvβ6 in alveolar epithelial cells also increases significantly. This upregulation of expression has been shown to be of great significance in promoting pulmonary fibrosis, which has also been confirmed in a mouse model of radiation-induced pulmonary fibrosis in response to monoclonal antibody inhibition of αvβ6.

[0013] High expression of αvβ6 in cancer is associated with a poor prognosis and can participate in the interaction between tumor cells and the extracellular matrix through the RGD sequence in fibronectin and collagen. Overexpression of this integrin is related to the transformation of epithelial cells into mesenchymal-like cells, which can promote the invasion and migration of tumor cells. Usually, in the leading edge of tumor invasion foci, the expression level of integrin is relatively high. There is also evidence that blocking αvβ6 signaling can prevent in vivo tumor progression in cancers such as pancreatic cancer, ovarian cancer, breast cancer, and colon cancer. In addition, studies have also shown that the activation of transforming growth factor-β by αvβ6 is an important pathway for immunosuppressive signals in various cancers. It is worth noting that TGF-β1 is usually bound to the latency-associated peptide (LAP) and exists in an inactive latent form (ProTGF-β1), and the LAP contains an RGD sequence that can be bound by αvβ6. The binding of LAP to αvβ6 can release and activate the latent TGF-β1. Therefore, αvβ6-positive cancer cells can be self-sufficient in growth signals. For example, in the study of cervical cancer patients, patients with positive αvβ6 expression had upregulation of the expression of four proliferation markers, namely P53, proliferating cell nuclear antigen, Ki-67, and TIPE2, to varying degrees.

[0014] A20FMDV2 polypeptide

[0015] The linear 20-mer peptide A20FMDV2 containing the amino acid sequence NAVPNLRGDLQVLAQKVART (SEQ ID NO:27) is considered to be an effective αvβ6 inhibitor in vivo. A20FMDV2 is derived from the polypeptide sequence of the foot-and-mouth disease virus envelope protein, and it mediates virus infection by binding to αvβ6. In this peptide, the sequence RGDLQVL is responsible for targeting αvβ6. This peptide has a high specificity for the αvβ6 integrin and minimal interaction with other RGD-recognizing integrins such as αvβ5 and αvβ3. In the interaction between the A20FMDV2 polypeptide and αvβ6, the Arg RGD (basic) side chain interacts with the carboxyl group on Asp218 of the αv subunit through a bidentate hydrogen bond, while the carboxyl group on Asp RGD (acidic) can interact with Mg in the metal ion-dependent adhesion site (MIDAS) of the β6 subunit 2+Ionic coordination. In addition, the LQVL sequence immediately following RGD can form a short α-helix, and a highly specific hydrophobic interaction can form between this secondary structure and the β6 subunit in the αvβ6 receptor, enabling the A20FMDV2 polypeptide to have high affinity and strong target specificity. However, the in vivo stability of this tracer is poor because studies have shown that it can be decomposed into various metabolites in animals, which may be disadvantageous for the transformation of this ligand into clinical applications. Summary of the Invention

[0016] In one aspect, the present application provides a peptide comprising or consisting of a peptide sequence having a first amino acid residue substitution and a second amino acid residue substitution relative to the sequence of SEQ ID NO:28 (NAVPNLRGDLQVLAQXVART, where X is K or N), wherein the first amino acid residue substitution is located at the position corresponding to positions 11 or 12 of SEQ ID NO:28, the second amino acid residue substitution is located at the position corresponding to positions 6, 14, or 15 of SEQ ID NO:28, and the first side chain of the first amino acid residue substitution crosslinks with the second side chain of the second amino acid residue substitution.

[0017] In one aspect, the use of the peptide of the present application in the preparation of a reagent for targeting αvβ6, particularly a tracer for αvβ6, more particularly an in vivo tracer for αvβ6, is provided.

[0018] In some embodiments, the peptide of the present application, for example, has a strong specific binding force to αvβ6 and a strong targeting ability to αvβ6. In some embodiments, the peptide of the present application has high stability. In some embodiments, the peptide of the present application has a strong specific binding force to αvβ6 and a strong specific binding force to cells.

[0019] The above technical effects of the peptide of the present application may be due to the peptide of the present application having a crosslinking across the sequence RGDL or the sequence L in the sequence RGDLQVL, making the sequence RGD therein less likely to be recognized and decomposed by enzymes such as proteases. Brief Description of the Drawings

[0020] Figure 1 It is the LC-MS / MS diagram of the peptide of SEQ ID NO:7 in the examples of the present application and the comparative example A20. Detailed Description of the Embodiments

[0021] To further elaborate on the technical means and effects adopted by the present application to achieve the predetermined purpose, the following, in combination with the accompanying drawings and preferred embodiments, details the specific embodiments, structures, features, and their effects according to the present application.

[0022] Terms and definitions

[0023] As used herein, the singular terms refer to one or more than one. For example, "element" or "an element" both refer to one element or more than one element. As used herein, the term "plurality" means at least two.

[0024] As used herein, the term "about" means approximate, in the range of about or near. When the term "about" is used in connection with a numerical range, it modifies the range by extending the boundaries above or below the provided value. Generally, the term "about" as used herein causes the value to vary up and down by 10% of the provided value. In one aspect, the term "about" means plus or minus 20% of the numerical value of the number it modifies. For example, "about 50%" means in the range of 45% - 55%. Numerical ranges mentioned herein by endpoints include all integers and fractions included within that range (e.g., "1 to 5" includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all integers and fractions are considered to be modified by the term "about".

[0025] As used herein, the terms "comprising", "including", or "containing", as non-exclusive or open-ended terms, are intended to mean that the combination (e.g., apparatus, composition, method, etc.) includes the recited elements (e.g., each unit of the apparatus, each component of the composition, the substantial steps of the method, etc.), but does not exclude other elements. As used herein, the term "consisting essentially of", when used to define compositions and methods, means excluding other elements that have any substantial influence on the combination for the stated purpose, but does not exclude other elements that do not substantially affect the basic and novel features of the invention. As used herein, the term "consisting of" means excluding other combinations of elements (units, components, substantial steps, etc.), but unless otherwise stated, does not mean excluding trace amounts of unavoidable impurities. Embodiments defined by each of these connecting terms are within the scope of the present invention. As a specific embodiment thereof, a technical solution disclosed including the terms "comprising", "including", or "containing" should also be considered as simultaneously disclosing the corresponding technical solutions including the terms "consisting essentially of" and "consisting of".

[0026] As used herein, the term "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items can be included alone, or any combination of two or more of the listed items can be included. For example, if a group, combination, or composition, etc., is described as including (or comprising) components A, B, C, and / or D, then the composition can include A alone; B alone; C alone; D alone; a combination of A and B; a combination of A and C; a combination of A and D; a combination of B and C; a combination of B and D; a combination of C and D; a combination of A, B, and C; a combination of A, B, and D; a combination of A, C, and D; a combination of B, C, and D; or a combination of A, B, C, and D.

[0027] As used herein, the term "amino acid" may be used interchangeably with "amino acid residue" and may refer to free amino acids and amino acid residues of peptides. It will be clear from the context in which the term is used whether it refers to free amino acids or residues of peptides. As described herein, "amino acid" is intended to include natural and synthetic amino acids and includes D- and L-amino acids. "Natural amino acid" refers to any one of the twenty standard L-amino acids (including glycine) that are typically found in naturally occurring peptides. As used herein, "D-" and "L-" amino acids, unless otherwise specified, are not intended to exclude amino acids that do not have chirality, such as glycine. "Unnatural amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or derived from natural sources. As used herein, "synthetic amino acid" also includes chemically modified amino acids, including but not limited to salts, amino acid derivatives (e.g., amides), and substituents. Amino acids contained in the peptides of the present invention can be modified by methylation, amidation, acetylation, or substitution with other chemical groups that can alter the circulatory half-life of the peptide without detrimentally affecting its activity, and particularly amino acids located at the C-terminus or N-terminus.

[0028] As used herein, the term "amino acid residue substitution" means that one amino acid residue has been substituted relative to a reference sequence. The definition herein "having an amino acid residue substitution of amino acid z at the position corresponding to position y of the sequence of SEQ ID NO: x relative to the sequence of SEQ ID NO: x" means that the peptide sequence described by this definition is different from the sequence of SEQ ID NO: x at least in the amino acid residue corresponding to position y of SEQ ID NO: x and is the amino acid residue z.

[0029] The term "amino acid analogue" refers to a molecule that is structurally similar to an amino acid and can replace an amino acid in the formation of a stapled peptide. Amino acid analogues include, but are not limited to, compounds that are structurally the same as the amino acids defined herein, but also contain one or more additional methylene groups between the amino and carboxyl groups (e.g., α-amino carboxylic acids), or replace the amino or carboxyl group with a similar reactive group (e.g., replace a primary amine with a secondary or tertiary amine, or replace a carboxyl group with an ester).

[0030] As used herein, the term "stapled peptide" refers to a peptide or its derivative that contains multiple amino acid residues linked by multiple peptide bonds, and at least one linker that forms a macrocycle, which forms a crosslink between a first amino acid residue and a second amino acid residue within the same molecule, thereby forming a macrocycle that includes the first amino acid residue, the second amino acid residue, and each amino acid between their positions in the sequence. The manner of preparing stapled peptides is known in the art, for example, Gregory L. Verdine, Gerard J. Hilinski, "Stapled Peptides for Intracellular Drug Targets." Methods Enzymol. 2012, 503, 3 - 33, WO 2010 / 033617, and WO 2010 / 011313, but not limited to these. The disclosures of these documents are incorporated herein by reference.

[0031] The "crosslink" in this article can be formed, for example, by directly covalently bonding the side chain of the first amino acid residue and the side chain of the second amino acid residue or by connecting through a specific moiety; for example, the crosslink can be formed by directly covalently bonding or connecting through a specific moiety between the side chains of crosslinkable amino acid residues with each other.

[0032] The term "crosslinkable amino acid" refers to an amino acid having a side chain containing a functional group capable of crosslinking with another amino acid through a covalent bond, such as, but not limited to, natural amino acids such as glutamic acid, aspartic acid, lysine, cysteine, etc., but not limited to these, and crosslinkable amino acids may also include non-natural amino acids.

[0033] As used herein, the term "linear peptide" at least includes a peptide having a sequence corresponding to that of a stapled peptide, but without forming a crosslink between the first amino acid residue and the second amino acid residue. As used herein, the term "pharmaceutical composition" refers to a composition containing at least one active ingredient, which is acceptable for studying a specific, effective result in mammals (such as, but not limited to, humans). Based on the needs of those skilled in the art, those of ordinary skill in the art will understand and know the techniques suitable for determining whether the active ingredient has the desired effective effect.

[0034] As used herein, "polynucleotide" refers to single-stranded or parallel and antiparallel strands of nucleic acid. Thus, a polynucleotide can be single-stranded or double-stranded nucleic acid. As used herein, "polypeptide" refers to a polymer composed of amino acid residues, their related naturally occurring structural variants, and synthetic non-naturally occurring analogs, which are linked by peptide bonds, their related naturally occurring structural variants, and synthetic non-naturally occurring analogs. As used herein, "synthetic peptide or polypeptide" refers to a peptide or polypeptide that is not naturally occurring. For example, the synthetic peptide or polypeptide can be synthesized using an automated polypeptide synthesizer.

[0035] As used herein, the notations at the bonding ends in the structural formula represent a linking bond. The linking bond can be to connect other parts of the polypeptide structure or to connect a substituent group, especially a simple substituent group such as hydrogen or methyl. For example, for the amide bond represented by the structural formula the carbon atom and the nitrogen atom can each be connected to other parts of the polypeptide structure; for the triazole moiety represented by the structural formula among the three linking bonds, two of them can each be connected to other parts of the polypeptide structure, and the remaining one is connected to a substituent group such as hydrogen or methyl.

[0036] As used herein, in the structural formula the representation of a double bond does not define its Z / E (cis / trans) configuration, regardless of how the other substituents at both ends of the double bond are represented in the structural formula.

[0037] Embodiments of the present application

[0038] In one aspect, the present application provides a peptide, especially a synthetic peptide, comprising or consisting of a peptide sequence having, and especially having only, a first amino acid residue substitution and a second amino acid residue substitution relative to the sequence of SEQ ID NO:28 and especially having an identity of more than 90%, wherein the first amino acid residue substitution is at a position corresponding to the 11th or 12th position of SEQ ID NO:28, the second amino acid residue substitution is at a position corresponding to the 6th, 14th, or 15th position of SEQ ID NO:28, and the first amino acid residue substitution and the second amino acid residue substitution are crosslinked through their side chains.

[0039] In some embodiments, the first amino acid residue substitution and the second amino acid residue substitution are located at positions corresponding to positions 12 and 15, positions 11 and 14, or positions 11 and 6 of SEQ ID NO:28, respectively. In some embodiments, the first amino acid residue substitution is located at the position corresponding to position 11 of SEQ ID NO:28. In some embodiments, the first amino acid residue substitution is located at the position corresponding to position 12 of SEQ ID NO:28. In some embodiments, the second amino acid residue substitution is located at the position corresponding to position 6 of SEQ ID NO:28. In some embodiments, the second amino acid residue substitution is located at the position corresponding to position 14 of SEQ ID NO:28. In some embodiments, the second amino acid residue substitution is located at the position corresponding to position 15 of SEQ ID NO:28. In some embodiments, the first amino acid residue substitution is located at the position corresponding to position 12 of SEQ ID NO:28 and the second amino acid residue substitution is located at the position corresponding to position 15 of SEQ ID NO:28. In some embodiments, the first amino acid residue substitution is located at the position corresponding to position 11 of SEQ ID NO:28 and the second amino acid residue substitution is located at the position corresponding to position 14 of SEQ ID NO:28. In some embodiments, the first amino acid residue substitution is located at the position corresponding to position 11 of SEQ ID NO:28 and the second amino acid residue substitution is located at the position corresponding to position 6 of SEQ ID NO:28.

[0040] In some embodiments, the crosslinking includes at least one covalent bond, aromatic ring moiety, or heterocyclic moiety between the first side chain and the second side chain. In some embodiments, the crosslinking includes at least one covalent bond between the first side chain and the second side chain. In some embodiments, the covalent bond is an amide bond, a disulfide bond, or an alkene bond. In some embodiments, the crosslinking includes at least one aromatic ring moiety between the first side chain and the second side chain. In some embodiments, the aromatic ring moiety includes a benzene ring moiety. In some embodiments, the crosslinking includes at least one heterocyclic moiety between the first side chain and the second side chain. In some embodiments, the heterocyclic moiety is a 5- to 6-membered ring moiety including 1–3 nitrogen atoms, particularly a 5- to 6-membered ring moiety including 1–3 adjacent nitrogen atoms and at least one double bond, more particularly a 1,2,3-triazole moiety, a pyrazoline moiety, or a dihydropyridazine moiety.

[0041] In some embodiments, the crosslinking includes a selection from the group consisting of Moieties of the group formed. In some embodiments, the crosslinking comprises a moiety or moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties. In some embodiments, the crosslinking comprises moieties.

[0042] In some embodiments, a crosslink is formed between the first amino acid residue substitution and the second amino acid residue substitution through side chains each comprising one selected from the group consisting of a carboxyl moiety, an amino moiety, a thiol moiety, an alkenyl moiety, an azide moiety, an alkynyl moiety, a tetrazole moiety, a 1,2,4,5-tetrazine moiety, and a cyclooctene moiety.

[0043] In some embodiments, a crosslink is formed between the first amino acid residue substitution and the second amino acid residue substitution via side chains each including one selected from the group consisting of a carboxyl moiety, an amino moiety, and a thiol moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including a carboxyl moiety and an amino moiety, a thiol moiety and a thiol moiety, a thiol moiety and an alkyne moiety, an alkene moiety and an alkene moiety, an azide moiety and an alkyne moiety, a tetrazole moiety or an alkene moiety, or a 1,2,4,5-tetrazine moiety and or a cyclooctane moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including a carboxyl moiety and an amino moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including a thiol moiety and a thiol moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including a thiol moiety and an alkyne moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including an alkene moiety and an alkene moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including an azide moiety and an alkyne moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including a tetrazole moiety or an alkene moiety. In some embodiments, a crosslink is formed between the first amino acid residue substitution or the second amino acid residue substitution via side chains respectively including a 1,2,4,5-tetrazine moiety and or a cyclooctane moiety.

[0044] In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is a crosslinkable amino acid, such as glutamic acid, aspartic acid, lysine, or cysteine. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is glutamic acid or aspartic acid. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is glutamic acid. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is aspartic acid. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is lysine. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is cysteine. In some embodiments, one of the first amino acid residue substitution and the second amino acid is glutamic acid or aspartic acid, and the other is lysine. In some embodiments, the first amino acid residue substitution is glutamic acid or aspartic acid, and the second amino acid is lysine. In some embodiments, the first amino acid residue substitution is lysine, and the second amino acid is glutamic acid or aspartic acid. In some embodiments, both the first amino acid residue substitution and the second amino acid are cysteine. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is an amino acid comprising an alkenyl moiety, an azide moiety, an alkynyl moiety, a 1,2,4,5-tetrazine moiety, or a cyclooctene moiety. In some embodiments, one of the first amino acid residue substitution and the second amino acid residue substitution is cysteine, and the other is an amino acid comprising an alkynyl moiety. In some embodiments, one of the first amino acid residue substitution and the second amino acid residue substitution is an amino acid comprising an alkenyl moiety, and the other is an amino acid comprising an alkenyl moiety. In some embodiments, one of the first amino acid residue substitution and the second amino acid residue substitution is an amino acid comprising an azide moiety, and the other is an amino acid comprising an alkynyl moiety. In some embodiments, one of the first amino acid residue substitution and the second amino acid residue substitution is an amino acid comprising a 1,2,4,5-tetrazine moiety, and the other is an amino acid comprising a cyclooctene moiety. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is a natural amino acid. In some embodiments, the first amino acid residue substitution or the second amino acid residue substitution is an unnatural amino acid.

[0045] In some embodiments, the amino acid at position 16 of SEQ ID NO:28 is lysine or asparagine. In some embodiments, the amino acid at position 16 of SEQ ID NO:28 is lysine. In some embodiments, the amino acid at position 16 of SEQ ID NO:28 is asparagine.

[0046] In some embodiments, the peptide comprises or consists of a peptide sequence as shown in any one of SEQ ID NO: 1–25. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in any one of SEQ ID NO: 21–25. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 3, 4, 6, 7, 9, 13, 14, 16, 19, 20, 21. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 1, 2, 4, 8, 10, 11, 12, 15, 17, 18. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 1. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 2. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 3. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 4. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 5. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 6. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 7. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 8. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 9. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 10. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 11. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 12. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 13. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 14. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 15. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 16. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 17. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 18. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 19. In some embodiments, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 20.In some embodiments, the peptide comprises the peptide sequence shown in SEQ ID NO: 21 or consists thereof. In some embodiments, the peptide comprises the peptide sequence shown in SEQ ID NO: 22 or consists thereof. In some embodiments, the peptide comprises the peptide sequence shown in SEQ ID NO: 23 or consists thereof. In some embodiments, the peptide comprises the peptide sequence shown in SEQ ID NO: 24 or consists thereof. In some embodiments, the peptide comprises the peptide sequence shown in SEQ ID NO: 25 or consists thereof.

[0047] In some embodiments, the affinity of the peptide for the αvβ6 receptor protein is greater than that of the peptide of any one of the sequences shown in SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.

[0048] In some embodiments, there is provided the use of the peptide of the present application in the preparation of a reagent targeting αvβ6, particularly a tracer of αvβ6, more particularly an in vivo tracer of αvβ6, or a reagent that specifically binds to cells expressing the αvβ6 receptor. In some embodiments, there is provided the use of the peptide of the present application in targeting αvβ6, particularly tracing αvβ6, more particularly in vivo tracing of αvβ6, or specifically binding to cells expressing the αvβ6 receptor. In some embodiments, there is provided the peptide of the present application for targeting αvβ6, particularly for tracing αvβ6, more particularly for in vivo tracing of αvβ6, or specifically binding to cells expressing the αvβ6 receptor. In some embodiments, there is provided a method for targeting αvβ6, particularly tracing αvβ6, more particularly in vivo tracing of αvβ6, or specifically binding to cells expressing the αvβ6 receptor, comprising administering the peptide of the present application. In some embodiments, the reagent targeting αvβ6, the tracer of αvβ6, or the in vivo tracer of αvβ6 is a drug for detecting cancer, diagnosing cancer, monitoring cancer progression, monitoring cancer treatment, or treating cancer. In some embodiments, targeting αvβ6, tracing αvβ6, or in vivo tracing of αvβ6 is for detecting cancer, diagnosing cancer, monitoring cancer progression, monitoring cancer treatment, or treating cancer.

[0049] Examples

[0050] I. Synthesis of A20 stapled peptide

[0051] Starting from the A20FMDV2 sequence (hereinafter referred to as "A20"), a series of αvβ6-targeting stapled peptides were designed and synthesized, and their affinity for the αvβ6 receptor protein was evaluated by ELISA experiments. The designed and synthesized αvβ6-targeting stapled peptides are shown in Table 1.

[0052] Table 1 αvβ6-targeted stapled peptides designed and synthesized in this application

[0053]

[0054] In the sequences of Table 1: The bold amino acids are different from the corresponding positions of the A20FMDV2 polypeptide; the underlined amino acids form the stapled peptide structure, that is, the amino acids at both ends of the underline are connected by bonds other than the main-chain peptide bond.

[0055] For the comparative example polypeptide of SEQ ID NO:26, see Ian I. Cardle et al. “Optimized serum stability and specificity of an αvβ6 integrin-binding peptide for tumor targeting” J. Biol. Chem. 2021, 296, 100657.

[0056] (I) Peptide synthesis: from the C-terminus to the N-terminus of the sequence.

[0057] Step 1. Weigh n equivalents of resin and put it into a reactor, add DCM (dichloromethane) to swell for half an hour. Then pump out the DCM, add 2n equivalents of the first amino acid in the sequence, and add 2n equivalents of DIEA (diisopropylethylamine) and an appropriate amount of DMF (dimethylformamide), and bubble with nitrogen for one hour. Then add about 5n equivalents of methanol and react for half an hour. Pump out the reaction solution and wash the resin with DMF and methanol.

[0058] Step 2. Add 2n equivalents of the second amino acid in the sequence, and 2n equivalents of HBTU (1-hydroxybenzotriazole tetramethyl hexafluorophosphate) and DIEA to the reactor, and bubble with nitrogen for half an hour. Wash off the liquid, detect with ninhydrin, and then cap with pyridine and acetic anhydride. Finally, wash and add an appropriate amount of deprotection solution to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, wash, and detect with ninhydrin.

[0059] Step 3. Repeat Step 2, each time adding different amino acids in the sequence in turn to synthesize the amino acids in the sequence onto the polypeptide in turn and perform the required modifications.

[0060] Step 4. After drying the resin with nitrogen, remove it from the reaction column and pour it into a flask. Then add a certain amount (about at a ratio of 10 mL of cleavage solution per gram of resin) of cleavage solution (composed of 95% TFA, 2% ethanedithiol, 2% triisopropylsilane, and 1% water) to the flask.

[0061] Step 5. Shake and filter off the resin to obtain a filtrate. Then add sufficient ether to the filtrate to precipitate the crude product. Centrifuge and wash to obtain the crude product of the sequence.

[0062] Step 6. The obtained crude product was purified by high performance liquid chromatography (HPLC), followed by lyophilization, and the molecular weight of the target product was confirmed by liquid chromatography-mass spectrometry (LC-MS).

[0063] (II) Evaluation of the affinity between the peptide and the αvβ6 receptor

[0064] The binding affinity of the polypeptides of the examples and comparative examples for the integrin αvβ6 recombinant protein was determined by ELISA (enzyme-linked immunosorbent assay).

[0065] 1. Preparation of A20-biotin

[0066] The polypeptide A20 of the comparative example was dissolved in a DMF solution before removing the ivDde protecting group on lysine in the sequence. The pH of the solution was adjusted to 8.0 - 9.0 with DIPEA. Biotin-NHS was added for the coupling reaction to obtain the A20-biotin polypeptide, which was purified by HPLC.

[0067] 2. Protein coating

[0068] Human αvβ6 protein was diluted with ELISA coating buffer to a final concentration of 2.5 μg / mL, and added to the well plate at 100 μL / well, and coated overnight at 4°C. Then the well plate was washed 6 times with 300 μL / well of phosphate buffered saline with Tween (PBST).

[0069] 3. Well plate blocking

[0070] 300 μL / well of blocking buffer (PBST solution containing 1% BSA) was added to the well plate and blocked at room temperature for 2 hours. Then the well plate was washed 6 times with 300 μL / well of PBST.

[0071] 4. Sample dilution

[0072] (1) Ligand molecule: A20-biotin, diluted with Tris buffered saline (TBS) to a concentration of 4 μM

[0073] (2) Positive control: A20, diluted with TBS to concentrations of 32, 16, 8, 4, 2, 1, 0.5 μM respectively

[0074] (3) Negative control: A20-Random (A20 with the original amino acid sequence scrambled), diluted with TBS to a final concentration of 40 μM

[0075] (4) Test polypeptides: Examples 1 - 20, diluted with TBS to concentrations of 200, 20, 2, 0.2, 0.02 μM respectively

[0076] 5. Add test samples

[0077] Add the sample in the amounts shown in Table 1 and shake the reaction at room temperature for 2 hours. Then wash the well plate 6 times with 300 μL / well of PBST.

[0078] Table 1

[0079]

[0080]

[0081] 6. Add the secondary antibody

[0082] Dilute streptavidin-HPR (1:100) with PBST and add 100 μL to each well. Shake the reaction at room temperature for 1 hour. Then wash the well plate 6 times with 300 μL / well of PBST.

[0083] 7. Add the substrate

[0084] Add 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) chromogenic solution to each well, immediately place the well plate in a microplate reader for incubation in the dark, read the OD value at a wavelength of 650 nm every 5 minutes for a total of 1 hour (13 detection points).

[0085] 8. Data processing

[0086] Process the data with Graphpad software and calculate the IC 50 value.

[0087] The IC 50 values of the examples are shown in Table 1. Table 1 shows that most of the examples have a stronger binding affinity for the integrin αvβ6 recombinant protein than Comparative Example A20.

[0088] II. Stability study

[0089] Use the in vitro constant temperature (37 °C) incubation method to study the in vitro stability of two polypeptides (1.0 μg / mL), Example SEQ ID NO:7 and Comparative Example A20, in rat plasma.

[0090] Dissolve the polypeptide in 1 mL of rat serum, and after in vitro constant temperature incubation for 3–24 h, determine the remaining percentage of the drug. Use LC-MS / MS to measure the peak areas of the drug prototype and its various metabolites, and calculate using the ratio of their peak areas instead of the drug concentration.

[0091] The results are as Figure 1 . Figure 1 This is the LC-MS / MS diagram of Example SEQ ID NO:8 and Comparative Example A20 of this application.

[0092] By Figure 1The main cleavage sites and metabolites of the two polypeptides of Example SEQ ID NO:8 and Comparative Example A20 can be confirmed. The cleavage sites are at P–N at positions 4–5 and R–G at positions 7–8 of the two sequences, as shown in Formula (I) and Formula (II).

[0093] Cleavage sites of A20

[0094] Cleavage sites of SEQ ID NO:8

[0095] The remaining drug (polypeptide) content was separated and identified by LC-MS.

[0096] Within 3 hours of incubation, both Example SEQ ID NO:8 and Comparative Example A20 were relatively stable. After 19 hours of incubation, only about 25% of the polypeptide prototype molecule of Comparative Example A20 remained, while about 37% of the stapled peptide prototype molecule of Example SEQ ID NO:8 remained. In particular, Figure 1 It shows that the proportion of the cleavage product at the R–G position of Example SEQ ID NO:8 is significantly less than that of Comparative Example A20, proving that the stapled peptide of the embodiment of the present application stabilizes the polypeptide molecular structure to a certain extent.

[0097] In addition, the in vitro stabilities of Example SEQ ID NO:21 and Comparative Example SEQ ID NO:26 were also compared, and the results are shown in Table 2.

[0098] Table 2

[0099]

[0100] The RGD motif is the key sequence for the polypeptide to recognize the integrin αvβ6 receptor. The cleavage between R–G will cause the polypeptide to lose the recognition and binding to the αvβ6 receptor. From the above experimental results, it shows that the stapled peptides of the present application, such as the cyclic peptides of SEQ ID NO:1–25, due to their formation of small cyclic peptide structures, reduce the cleavage degree at this site to a considerable extent compared with other macrolide stapling peptide cyclization strategies. Therefore, the in vitro stabilities of these cyclic peptides are all improved. In particular, the cyclic peptides of Example SEQ ID NO:21–25 form a six-membered cyclic peptide through the stapling peptide strategy, further reducing the recognition and cleavage of the RGD motif by proteases, making the in vitro stabilities of these cyclic peptides more significantly improved. This not only improves the overall stability of the polypeptide molecule, but also ensures the integrity of the key site and enhances the stable targeting ability in vivo.

[0101] III. Cell binding experiment

[0102] The human pancreatic cancer cell line BxPC-3 was cultured normally at 37°C under 5% CO2. Before the cell binding experiment, appropriate trypsin digestion solution was used for trypsin digestion, and the cells were counted and grouped as shown in Table 3, with 2×10 5 cells in each group.

[0103] Table 3

[0104] Grouping Primary antibody (1 μM) Blocking agent (100 μM) Secondary antibody (12 ng) Positive rate 1 (negative blank control) / / Streptavidin-PE 3.3％ 2 (positive blank control) A20-biotin / Streptavidin-PE 84.4％ 3 (positive control 1) A20-biotin A20 polypeptide Streptavidin-PE 1.9％ 4 (negative control) A20-biotin A20-Random Streptavidin-PE 80.7％ 5 (experimental group) A20-biotin Polypeptide of SEQ ID NO:4 Streptavidin-PE 0.3％ 6 (positive control 2) A20-biotin TGF-β3 (1 μg) Streptavidin-PE 0.3％

[0105] The cells were treated according to the methods of each group in the above table.

[0106] First, 50 μL of primary antibody and 50 μL of blocker were added simultaneously, and the mixture was incubated on ice for 120 min, then centrifugally washed 3 times with 500 μL of PBS. Then, 50 μL of secondary antibody (0.06 μg / 10 6 cells) was added, and the mixture was incubated on ice for 60 min, then centrifugally washed 3 times with 500 μL of PBS. Next, flow cytometry was performed to detect the fluorescence signal in the channel with an emission wavelength of 575 nm and an excitation wavelength of 488 nm. A total of 5000 events were collected for each group, and the binding of the polypeptide of Example SEQ ID NO:4 and the polypeptide of Comparative Example A20 to BxPC-3 cells was evaluated by detecting the positive cell ratio.

[0107] The experimental results are shown in Table 2.

[0108] The results showed that the biotinylated polypeptide of the comparative example (A20-biotin) could specifically bind to BxPC-3 cells expressing the αvβ6 receptor, with a positive rate of 84.4%. This binding could be blocked by itself (A20), the polypeptide of the example (SEQ ID NO:4 polypeptide), and the natural ligand TGF-β3 of the αvβ6 receptor, but could not be blocked by the polypeptide with its original amino acid sequence scrambled (A20-Random), indicating that this binding was specific. Among them, the positive rate of the SEQ ID NO:4 polypeptide group was 0.3%, which was significantly lower than 1.9% of the A20 polypeptide, indicating that the SEQ ID NO:4 polypeptide had stronger specific binding ability compared to the A20 polypeptide.

[0109] The above embodiments are only the preferred embodiments of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and substitutions made by those skilled in the art based on the present application belong to the scope of protection required by the present application.

Claims

1. A peptide, characterized in that, it comprises or consists of a peptide sequence having a first amino acid residue substitution and a second amino acid residue substitution relative to the sequence of SEQ ID NO:28, wherein, the first amino acid residue substitution is located at the position corresponding to the 11th or 12th position of SEQ ID NO:28, the second amino acid residue substitution is located at the position corresponding to the 6th, 14th, or 15th position of SEQ ID NO:28, and the first side chain of the first amino acid residue substitution crosslinks with the second side chain of the second amino acid residue substitution.

2. The peptide according to claim 1, characterized in that, the first amino acid residue substitution and the second amino acid residue substitution are respectively located at the positions corresponding to the 12th and 15th positions of SEQ ID NO:28; the 11th and 14th positions; or the 11th and 6th positions of SEQ ID NO:28; Optionally, the first amino acid residue substitution and the second amino acid residue substitution are respectively located at the positions corresponding to the 12th and 15th positions of SEQ ID NO:28; Optionally, the first amino acid residue substitution and the second amino acid residue substitution are respectively located at the positions corresponding to the 11th and 14th positions of SEQ ID NO:28; Optionally, the first amino acid residue substitution and the second amino acid residue substitution are respectively located at the positions corresponding to the 11th and 6th positions of SEQ ID NO:

28.

3. The peptide according to claim 1, characterized in that, the crosslinking comprises at least one covalent bond, aromatic ring moiety, or heterocyclic moiety between the first side chain and the second side chain; Optionally, the covalent bond is an amide bond, disulfide bond, or alkene bond; Optionally, the aromatic ring moiety comprises a benzene ring moiety; Optionally, the heterocyclic moiety is a 5- to 6-membered ring moiety containing 1 - 3 nitrogen atoms, particularly a 5- to 6-membered ring moiety containing 1 - 3 adjacent nitrogen atoms and at least one double bond, more particularly a 1,2,3-triazole moiety, pyrazoline moiety, or dihydropyridazine moiety; Optionally, the crosslinking includes a moiety selected from the group consisting of ; Optionally, the crosslinking includes partial or portions.

4. The peptide according to claim 1, characterized in that, the crosslinking between the first amino acid residue substitution and the second amino acid residue substitution is formed through side chains each comprising one selected from the group consisting of a carboxyl moiety, amino moiety, thiol moiety, alkenyl moiety, azide moiety, alkynyl moiety, tetrazole moiety, 1,2,4,5-tetrazine moiety, and cyclooctene moiety; Optionally, the crosslinking between the first amino acid residue substitution and the second amino acid residue substitution is formed through side chains each comprising one selected from the group consisting of a carboxyl moiety, amino moiety, and thiol moiety.

5. The peptide according to claim 1, characterized in that, the first amino acid residue substitution is glutamic acid, aspartic acid, lysine, or cysteine; and / or the second amino acid residue substitution is glutamic acid, aspartic acid, lysine, or cysteine.

6. The peptide according to claim 1, characterized in that, one of the first amino acid residue substitution and the second amino acid residue substitution is glutamic acid or aspartic acid, and the other is lysine.

7. The peptide according to claim 1, wherein both the first amino acid residue substitution and the second amino acid residue substitution are cysteine.

8. The peptide according to claim 1, wherein the peptide comprises or consists of a peptide sequence as shown in any one of SEQ ID NO: 1–25; optionally, the peptide comprises or consists of a peptide sequence as shown in SEQ ID NO: 3, 4, 6, 7, 9, 13, 14, 16, 19, 20, 21.

9. Use of the peptide according to any one of claims 1 to 8 in the preparation of a reagent targeting αvβ6, in particular a tracer of αvβ6, more particularly an in vivo tracer of αvβ6, or a reagent that specifically binds to cells expressing the αvβ6 receptor.

10. The use according to claim 9, wherein the targeting of αvβ6, the tracing of αvβ6, or the in vivo tracing of αvβ6 is for detecting cancer, diagnosing cancer, monitoring cancer progression, monitoring cancer treatment, or treating cancer.

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